Plant Extracts as a Source of Natural Products with Potential Antimalarial Effects: An Update from 2018 to 2022

Malaria kills more than 500,000 people yearly, mainly affecting Africa and Southeast Asia. The disease is caused by the protozoan parasite from the genus Plasmodium, with Plasmodium vivax and Plasmodium falciparum being the main species that cause the disease in humans. Although substantial progress has been observed in malaria research in the last years, the threat of the spread of Plasmodium parasites persists. Artemisinin-resistant strains of this parasite have been reported mainly in Southeast Asia, highlighting the urgent need to develop more effective and safe antimalarial drugs. In this context, natural sources, mainly from flora, remain underexplored antimalarial spaces. The present mini-review explores this space focusing on plant extracts and some of their isolated natural products with at least in vitro antiplasmodial effects reported in the literature comprising the last five years (2018–2022).


Introduction
Malaria is still a worldwide public health problem, with an estimated 247 million cases and 619,000 deaths globally in 2022, principally affecting children in the African continent. Malaria primarily affects the intertropical region, where appropriate climatic and geographical conditions are suitable for the spread of the female Anopheles mosquito, the vector of the disease. The most prevalent species are Plasmodium falciparum and Plasmodium vivax, and the most severe form of the disease is caused by P. falciparum [1]. The rise of antimalarial resistance, mainly in Southeast Asia, due to the indiscriminate use of antimalarials and incomplete malaria drug treatment demonstrates the urgent need for the development of better and more efficacious drugs against the disease [2]. Although malaria prevention has shown considerable advances [3], the lack of an effective vaccine against the disease continues to increase the number of deaths in the African continent. In 2022, a ground-breaking malaria vaccine was prequalified by the WHO for the prevention of P. falciparum infections. This vaccine, named RTS, S/AS01 (Mosquirix) and developed in cooperation with the private pharmaceutical company GSK, was recommended by the WHO for the immunization of children from five months of age living in moderate-to high P. falciparum transmission regions in Africa. However, the effectiveness of this vaccine is not enough to completely halt the spread of severe malaria cases worldwide [4].
Despite the substantial public and private financial provision for malaria prevention resources and considerable scientific progress in the cellular, molecular, and clinical areas, chemotherapy remains a key tool for controlling the disease [5]. Malaria treatment has become increasingly complex, owing to the spread of artemisinin-resistant strains of Plasmodium. Recently, P. falciparum resistance to artemisinin derivatives has been detected in Southeast Asia [6,7]. As for P. vivax, the most important species on the African

Methodology for Literature Selection and Previous Literature
For this mini-review, a database search was performed with Digital Science's Dimensions platform (Dimensions AI), which also covers the PubMed database. The combination of keywords "Plasmodium" and "plant extract" in the title or abstract was used in the searches. The period covered is from 2018 to 2022. The search was initially conducted on 26 January 2023, but a few articles published after this date were also added due to the match of the keyword combination. The criteria for selecting the articles were primarily based on experimental data with a plant extract antiplasmodial activity less than 10 µg/mL. The articles were divided into two groups: those reporting natural products (divided into ethnobotanical studies and plant extracts) and those covering studies of isolated bioactive substances and possible pharmacological targets.
We have also analyzed recent reviews that referred to the use of medicinal plants as antimalarials [18][19][20][21][22][23]. They provide a thorough overview of the scientific literature with respect to the antimalarial effects of traditionally used medicinal plants, some of them mainly covering plants found in African countries, such as Ethiopia [20,22,23]. Another example is the review from Noronha and colleagues [21], which provides a broad revision of the literature concerning plants having antimalarial and antipyretic activity and the practical challenges of using these medicinal plants. In another review, Pan and collaborators [18] provide a comprehensive review covering more than 2000 plant extracts, including marine plants, and the discovery of 175 antimalarials from plants in the covered period of 2001-2017. The present mini-review differs from the cited previous literature, since it aims to provide an update on plant extracts and its most promising isolated compounds tested against Plasmodium falciparum, without restrictions to specific endemic plant species, and covering the last five years of scientific literature, as mentioned above.

Omics-Based Strategies for Targeted Isolation of Natural Products and Discovery of New Potential Drugs
Natural products play a key role in discovering new chemical entities [24], and plant extracts generally contain diverse chemical mixtures with different metabolites. Isolation of each chemical entity from these mixtures requires the use of combined analytical techniques, since the bioactive compounds are found in low concentrations and can also work in a synergistic mode. One challenging problem in the field of natural products is the identification of the mechanism of action (MOA) for small molecules. Target identification is crucial for compound optimization and further steps in the drug discovery process. However, it is also known that many drugs interact with different protein targets [25], which require broader target identification methods. In this sense, chemical proteomics may provide insights into the mechanism of action for small molecules. In chemical proteomics, the combination of chemistry, cellular biology, and mass spectrometry allows the generation of large data sets, with the possibility of understanding potential off-targets and how to generate a better lead compound. A crucial step in a chemical proteomics experiment is the probe design and synthesis, which depend on different probes, comprehensively revised by Chen and colleagues [25]. In chemical proteomics approaches, the natural products should be either chemically modified with an enrichment moiety (activity-based protein profiling) [26,27], linked to a resin, or conjugated with photoactivatable crosslinkers [28]. These approaches have been combined with quantitative label-free or label-based proteomics strategies to identify protein targets in complex biological systems. One drawback resides in the knowledge of structure-activity relationships, since chemical modifications can induce activity loss and binding to the wrong target.
The deconvolution of a protein target in an unbiased way has also been achieved using other approaches that explore protein stability upon small molecule binding. Protein stability can be challenged by raising the temperature, which will induce denaturation, aggregation, and precipitation. Proteins binding to a specific small molecule will respond differently to temperature changes. Thermal Proteome Profiling (TPP), cellular thermal shift assay coupled with mass spectrometry (CETSA ® ) [29], and Proteome Integral Solubility Alteration (PISA) [30] are based on thermal shifts. These assays can be performed in vitro, intact cell lines or cell lysates, and in vivo, in tissues and biofluids. MS-CETSA was applied to identify P. falciparum purine nucleoside phosphorylase as the target of quinine [31]. Another assay, termed drug affinity responsive target stability (DARTS), measures the resistance of a protein to hydrolysis by a broad specificity protease in the presence of a known small molecule [32]. The same principle was exploited in the limited proteolysis/LiP-small molecule mapping (LiP-SMap) [33]. Protein stability upon interaction with a small protein was also probed using the rate of methionine oxidation upon incubation with a denaturing agent. This assay was termed stability of proteins from rates of oxidation (SPROX) [34]. Recently, the resistance of a protein binding to a small molecule was probed using organic solvents. In this assay, termed solvent proteome profiling (SPP), the association of proteins with small molecule makes them more or less susceptible to solvent-induced denaturation [35]. The small molecule does not need to be modified in these assays, and its target(s) can be identified using a purified protein or a complex proteome. One drawback of these strategies is the solubility of membrane proteins. Indeed, the need to work with soluble proteins in their native state limits their applicability to multipass transmembrane proteins. Combining mass spectrometry-based proteomics strategies with these assays allows the identification of unknown targets.
Metabolomics has evolved to allow the analysis of thousands of metabolites from natural extracts [12], and it has been used to characterize metabolites of less than 1.5 kDa, such as amino acids and lipids, based mainly on nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. It has already been used for the identification of drug targets, as well: early metabolomics studies permitted, for example, the development of FDAapproved drugs enasidenib (Idhifa ® ) and ivosidenib (Tibsovo ® ) for the treatment of acute myeloid leukemia [36][37][38][39].
Moreover, the application of metabolomics in the screening of natural product extracts via hyphenated techniques allows detailed structural characterization [40]. It also permits implementing databases that can be used to discover new bioactive natural products [41].
Recent developments in analytical chemistry include dereplication and high content screening (HCS). Dereplication refers to a process for the fast identification of known secondary metabolites in natural products, in which MS-based methods are key to the success of that process [42][43][44], and HCS methods are those that combine automated microscopy with living cell-based assays [45][46][47][48], which allows, e.g., the screening of plant extract libraries and subsequent selection of the best hits based on the phenotypic screening results. However, the isolation of chemical compounds from crude natural extracts remains a major challenge in the field [42]. The extraction method determines the chemical compound classes present in the purified fractions. Based on the polarity of the molecules present in the extract, using different organic solvents can selectively isolate a range of different compounds, directly affecting phytochemical isolation and yield. The combination of solvents would be a strategy to improve the extraction of compounds with different polarities [49], as illustrated in Figure 1. After identifying a crude extract with promising pharmacological activity, the extract is fractionated and guided by bioactivity until pure bioactive compounds are isolated [50].
Other limitations of crude extraction steps include the presence of known natural product molecules, chemical compounds without drug-like characteristics, and insufficient amounts of natural products for characterization [47]. These technical challenges can be addressed by the development of new methods using dereplication, extraction, and prefractionation of extracts [51]. Another approach is the use of metabolomics and genomics, in which data generated by molecular networks are analyzed and common characteristics of the natural extract composition are determined. The low yield in the extraction process and the structural elucidation of the chemical compounds are other aspects to be addressed in the field [47]. Bioinformatics tools and genomic engineering may also be helpful. Tandem mass spectrometry (MS/MS) libraries allow fast, reproducible, and accurate analysis of thousands of MS/MS spectra. Because there is a need for reliable information on MS/MS, In addition, the web platform Global Natural Product Social Molecular Networking allows the generation of a molecular network in which molecules are grouped based on their fragmentation pattern of similarity and similarity in the identification of metabolites [52].
nomics, in which data generated by molecular networks are analyzed and common char-acteristics of the natural extract composition are determined. The low yield in the extraction process and the structural elucidation of the chemical compounds are other aspects to be addressed in the field [47]. Bioinformatics tools and genomic engineering may also be helpful. Tandem mass spectrometry (MS/MS) libraries allow fast, reproducible, and accurate analysis of thousands of MS/MS spectra. Because there is a need for reliable information on MS/MS, different databases, including NIST, MassBank, MoNA European MassBank, LipidBlast, Wiley MSforID, and METLIN, are available for this purpose. In addition, the web platform Global Natural Product Social Molecular Networking allows the generation of a molecular network in which molecules are grouped based on their fragmentation pattern of similarity and similarity in the identification of metabolites [52].

Novel Antimalarials Derived from Plant Extracts and Isolated Natural Products
Antiplasmodial natural products are organized into families of promising extracts, and isolated compounds are organized in classes, such as alkaloids, terpenes, and polyphenols, with subclasses when applicable. The in vitro antiplasmodial activities described in this review were determined using various strains of P. falciparum with different drug sensitivities. The chloroquine-sensitive strains were 3D7, NF54, D6, HB3, D10, and TM4/8.2. Other strains used were the chloroquine-resistant strains Dd2, FcB1, and Pf INDO, as well as the multiresistant strains K1, W2, FCR3, and K1CB1. Different assays were used to determine in vitro antiplasmodial activity of the compounds. The most cited methods are the radioactive hypoxanthine incorporation assay, the colorimetric enzymelinked immunosorbent assay (ELISA) that measures P. falciparum lactate protein dehydro-

Novel Antimalarials Derived from Plant Extracts and Isolated Natural Products
Antiplasmodial natural products are organized into families of promising extracts, and isolated compounds are organized in classes, such as alkaloids, terpenes, and polyphenols, with subclasses when applicable. The in vitro antiplasmodial activities described in this review were determined using various strains of P. falciparum with different drug sensitivities. The chloroquine-sensitive strains were 3D7, NF54, D6, HB3, D10, and TM4/8.2. Other strains used were the chloroquine-resistant strains Dd2, FcB1, and Pf INDO, as well as the multiresistant strains K1, W2, FCR3, and K1CB1. Different assays were used to determine in vitro antiplasmodial activity of the compounds. The most cited methods are the radioactive hypoxanthine incorporation assay, the colorimetric enzyme-linked immunosorbent assay (ELISA) that measures P. falciparum lactate protein dehydrogenase (pLDH), DNA-based fluorometric method using Pico Green Assay (SYBR), Malstat reagent, and microscopy. Regardless of the in vitro assay method, in this review, the inhibitory concentration range and extracts with half maximal inhibitory concentrations (IC 50 ) ≤ 10 µg/mL were considered active [53]. Only isolated compounds with IC 50 ≤ 10.0 µM were considered for further studies [54][55][56]. Based on these criteria, from the selected literature, a total of twenty isolated natural products derived from three different plant species were found to inhibit Plasmodium parasite proliferation in vitro (Table 1 and Figure 2). Phytochemical investigation of aqueous and ethanolic leaf extracts of Azadirachta indica (Meliaceae) showed inhibition of P. falciparum with IC 50 values of 7.4 µg/mL and 8.6 µg/mL, respectively. A study on the in vitro antimalarial activity of extracts from indigenous plant species in Kebbi State justifies the traditional use of the plant in malaria treatment, and further research may help identify and characterize the active principles of these plants [57].
The Homalolepis suffruticosa of the Simaroubaceae family is known for its popular use in treating malaria. Homalolepis Turcz. is a neotropical genus named Quinquina de Cayenne and represents one of the many drugs called 'falsa quina' (fake quine), which is used to treat malaria in South and Central America. In a bioassay-guided assay [58], the methanol extract of the roots of H. suffruticosa showed high in vitro antiplasmodial activity with an IC 50 of 1.88 µg/mL in moderate cytotoxic HepG2 (CC 50 = 41.93 µg/mL) and an SI = 22.30.
Goniothalamus lanceolatus (Annonaceae), an endemic plant from Borneo, is traditionally used in Malaysia to alleviate fever, as part of antimalarial treatment by folk medicine. Upon screening, methanol extract from roots exhibited potent antiplasmodial activity against both P. falciparum 3D7 (IC 50 = 2.7 µg/mL, SI = 140) and K1 strains (IC 50 = 1.7 µg/mL, SI = 236) [59]. The Annonaceae family is the largest family in Magnoliales order, comprising more than 100 genera and about 2400 species [76]. The large number of genus/species, with some of them reported in the literature as having antimalarial effects, such as those mentioned above for Goniothalamus lanceolatus, may also suggest further thorough investigation of potential antimalarials in that family [77].

Alkaloids: Potential Antimalarial Activity
Bioassay-guided fractionation of Hypoestes forskaolii (Vahl) R.Br. produced a phenanthroquinolizidine alkaloid, the isolated compound 15β-hydroxycryptopleurine-N-oxide (17), which has antiplasmodial activity. The chloroquine-resistant (K1) and chloroquine-sensitive (FCR3) P. falciparum strains were very sensitive to the antimalarial activity of 15β-hydroxycryptopleurine-N oxide, with IC50 values of 6.11 and 5.13 nM, respectively, while using chloroquine as the positive control [80]. A total of 15 mg was obtained from the plant, and a combination of techniques were used to purify the compound: silica gel column chromatography, high performance liquid chromatography (HPLC), and NMR (H, C, and two-dimensional).
From Cissampelos pareira (Linn) (Menispermaceae), a plant traditionally used to treat various diseases in India, a root ethyl acetate fraction with the most promising compound hayatinine (12), a bisbenzylisoquinoline alkaloid, was isolated (a total of 23 mg), having IC50 values of 0.41 μM (INDO) and 0.509 μM (3D7) [81]. Chloroquine was used as the positive control. Silica gel column chromatography, HPLC, NMR (H, C, and twodimensional), and liquid chromatography coupled to mass spectrometry (LC/MS) were utilized for this alkaloid.
The phytochemical investigation of Aconitum heterophyllum (Wall. ex Royle) (Ranunculaceae) roots led to the identification of 2-O-cinnamoyl hetisine, a diterpenoid alkaloid in the chloroform fraction of the plant [82]. Antiplasmodial in vitro studies indicated an IC50 of 1.92 μM (INDO) and an IC50 10.8 μM (3D7). The yield of this compound was very low: from 2.6 g of chloroform fraction, only 16 mg of this hetisine were obtained.
The tazopsine alkaloid (compound 10 in Figure 2), isolated from Strychnopsis thouarsii Baill. (Menispermaceae), an endemic Malagasy plant, showed antiplasmodial activity and an of IC50, 7.88 μM against the NF54 strain, as well as a value of 4.07 μM against the 3D7 strain [83]. However, in P. yoelii infected mice, it was considered non-prophylactic, with 70% protection at a 100 mg/kg dose.

Terpenoids: Potential Antimalarial Activity
Sesquiterpene lactones have emerged as good starting points in the search for transmission-blocking drugs, with several studies demonstrating their potential. The most widely investigated member of this class of compounds is artemisinin, along with its derivatives. Greve and collaborators reported [84] that a triterpene derivative, compound rel-(16S,20S)-dihydroxydammar-24-en-3-one (16), had the most promising antimalarial activity. For this triterpene derivative, silica gel column chromatography, HPLC, NMR (H, C, and two-dimensional), and LC/MS were used.  Petasites japonicus, an herb of the family Asteraceae, has been used to treat several human diseases. In Asian countries, the traditional use of P. japonicus is based on its many recognized pharmacological activities, including analgesic, anti-inflammatory, bactericidal, and antimalarial properties. A phytochemical study showed that the ethanolic extract from P. japonicus leaves significantly inhibited the parasite with IC 50 values of 8.48 µg/mL and 7.83 µg/mL against strains 3D7 (CQ-sensitive) and Dd2, respectively [61]. The antiplasmodial activity of P. japonicus was also tested in vivo with P. berghei ANKA infected mice. The ethanolic extract of P. japonicus in a dose of 200 mg/kg lowered the parasitemia after four days of infection with a decrease percentage in the range from 65.21% to 97.14% [61].
In another study, the medicinal plant Senna occidentalis was evaluated for its phytochemical and activity against P. falciparum 3D7. The methanol extracts of the leaves and seeds of S. occidentalis (L.) (Fabaceae) (Leguminosae) showed significant activity against P. falciparum 3D7 with IC 50 values of 12.19 µg/mL and 6.82 µg/mL, respectively [62]. In Kenya, the roots of the plant S. occidentalis are traditionally used for malaria treatment by the Luhya, Digo, and Duruma communities: the roots of the plant are decocted and taken orally three times a day over the course of three to four days. The methanolic extracts of S. occidentalis show antiplasmodial activity with a calculated IC 50 of 1.76 µg/mL against 3D7 strain. No cytotoxicity was observed against a Vero cell line (CC 50 247, IS of 140). The methanolic extract was also tested in vivo (P. berghei ANKA), showing an antiplasmodial effect of 200 mg/kg and effective dose (DE 50 ) of 34.13 ± 5.14 mg/kg [78].
Plants of the genus Nauclea (Rubiaceae) are frequently used as folk medicines for the treatment of various diseases. Antimalarial activity of the hexane (IC 50 = 1.93 µg/mL) and methanol (IC 50 = 3.91 µg/ mL) fractions of Nauclea orientalis leaves against 3D7 P. falciparum was observed [63].
According to a previous investigation [64], the aqueous extracts of the leaves of the antimalarial Alchornea cordifolia (Euphorbiaceae) exhibited moderate activities against NF54 (chloroquine-sensitive parasite; IC 50 of 5.8 µg/mL), CamWT_C580Y (artemisinin-sensitive parasite; IC 50 17.8 µg/mL), and IPC 4912 (artemisinin-resistant parasite; IC 50 15.8 µg/mL). The results of this study support the traditional use of the species A. cordifolia in Ghana as a folk remedy for treating malaria.
The Helianthus annuus (Asteraceae) plant is traditionally used as a medicinal remedy for several diseases, including malaria, in Oro-oro Ombo, Malang, East Java, and Indonesia.
The ethanol extract of the roots and leaves of the species H. annuus exhibited antimalarial activity, with IC 50 values of 2.3 and 4.3 µg/mL, respectively, against P. falciparum 3D7 [65]. The in vivo antiplasmodial effects of the ethanolic extract from H. annuus were also evaluated: 100 mg/kg with DE 50 of 63.6 ± 8.0 mg/kg and 59.3 ± 13.2 mg/kg [65].
In Côte d'Ivoire, several traditional pharmacopeia plants, such as Pericopsis laxiflora and Harungana madagascariensis, have been used by various traditional practitioners for malaria treatment. The aqueous extract of the bark of H. madagascariensis (Hipperiacaceae) showed antiplasmodial activity, with an IC 50 value of 6.16 µg/mL against P. falciparum NF54 [66]. The methanolic extracts of P. laxiflora (Euphorbiaceae) showed the best antiplasmodial activity, with an IC 50 value of 7.44 µg/ mL against K1 [66].
Pleiocarpa, which belongs to the Apocynaceae family, is native to tropical Africa and is distributed from Senegal to Tanzania and Zimbabwe. Medicinal plants of this genus are well known for their use in the traditional treatment of pain, fever, and malaria. A study [67] investigated the dichloromethane/methanol (1:1) extract of Pleiocarpa bicarpellata roots, which showed strong antiplasmodial activity (IC 50 = 3.5 µg/mL) against P. falciparum NF54. Negative cytotoxicity against L6 cells was observed.
Another recent study [58] on the Psychotria plant, which is native to Amazonia and Atlantic Forest biomes in Brazil, evaluated the antiplasmodial effect of the acid-base extracts of Psychotria apoda and P. colorata (Rubiaceae), which showed 90% inhibition of the parasite P. falciparum W2.
Andrographis paniculata is a tropical medicinal plant belonging to the Acanthaceae family. It is traditionally used for medicinal purposes in Hong Kong, Philippines, Taiwan, China, India, Malaysia, Indonesia, and many South Asian countries. The chloroform extract of whole plants of Andrographis paniculata exhibited antimalarial activity with IC 50 values of 6.36 µg/mL and 5.24 µg/mL against strains 3D7 and K1, respectively, with no evidence of significant cytotoxicity against a mammalian cell line (CC 50 > 100 µg/mL) [69].
Chaniad and collaborators [70] investigated the plant Globba malaccensis (Zingiberaceae), which is native to Nakhon Si Thammarat Province, Thailand. The ethanolic extract of G. malaccensis rhizomes showed promising antimalarial activity against P. falciparum K1 (IC 50 = 1.50 µg/mL), with less toxicity to Vero cells (CC 50 > 80 µg/mL). The ethanolic extract of this plant exhibited a considerable reduction in P. berghei parasitemia (murine model). A maximum suppressive effect of this extract (60.53%) was observed with the highest administered dose (600 mg/kg).
In Indonesia, the plant Sanchus arvensis L. (Asteraceae) is a traditional medicinal plant employed for malaria treatment. The ethyl acetate fraction obtained from leaves of S. arvensis showed in vitro antiplasmodial activity, with an IC 50 of 2.92 µg/mL (3D7 strain), and it did not show cytotoxicity against human hepatic cells (Huh7it-1), with an SI of 97.03 [71]. The fraction was also tested in vivo (mice infected with P. berghei ANKA), showing an antiplasmodial activity below 100 mg/kg/day and ED 50 46.31 ± 9.36 mg/kg. Chloroquine and chloroquine diphosphate were the positive controls for the in vitro studies, and DMSO served as the negative control.
The plant Terminalia arjuna (Roxb. ex DC.) (Wight and Arn.), known as "arjuna" or "Sa-mor-tes" in Thailand, has been traditionally used for various human diseases, given its antibacterial, antitumoral, and antiallergic effects. Against P. falciparum (strain K1), in vitro results showed an IC 50 of 4.05 µg/mL [72]. In Vero cells, the CC 50 was higher than 200 µg/mL, an indication of low cytotoxicity. For the in vitro study, the authors employed artesunate as a positive control and DMSO as a negative control. The in vivo study with aqueous extract of the T. arjuana fruits demonstrated a suppression of 67.95% of Plasmodium parasites at a dose of 600 mg/kg (P. berghei ANKA, murine model) [72]. Another plant species from the same genus, Terminalia bentzoe (L.) L.f. (Combretaceae) [73], was tested against P. falciparum (Dd2 strain), with an IC 50 of 6.7 µg/mL and low cytotoxicity in Vero cells and CC 50 207.03 µg/mL.
Another plant with potential antimalarial properties is Bridelia atroviridis Müll. Arg., a bush from Euphorbiaceae family that is found in equatorial Africa. This plant has been used in traditional medicine to treat various diseases, such as diabetes, gonorrhea, and fungal infections. Djouwoung and colleagues [74] have shown the antiplasmodial activity of hydroethanolic bark extract of Bridelia atroviridis Müll. Arg., exhibiting an in vitro antiplasmodial activity, with an IC50 of 8.08 µg/mL against the Dd2 strain. It also presented low cytotoxicity (CC 50 > 100 µg/mL). The in vivo tests were performed in P. berghei NK 65 infected rats, resulting in a calculated ED 50 of 89 mg/kg.
In Nigeria, the plant species Annickia affinis (Exell) (Versteegh and Sosef) from Annonaceae family is traditionally used for the treatment of fever and malaria. Decoction with water for this plant resulted in IC 50 1.49 µg/mL (3D7 strain), while the CC 50 was above 200 µg/mL [75]. In the in vivo test of infected mice with P. berghei ANKA, the A. affinis methanol extract at an oral dose of 100 mg/kg suppressed parasite growth by 32.46%. It was also investigated whether the plant has synergism with other plants as well, such as secondary metabolites from the mixture of plants named hepta-herbal Agbo-iba [75].
In another recent study, Ledoux and colleagues [79] investigated the potential antimalarial effects of the leaves of Casearia coriacea Vent. (Salicaceae), a plant endemic to the Mascarene Islands. Clerodane diterpenes were isolated from dichloromethane fraction and tested against the Plasmodium parasite: caseamembrin T, corymbulosine I, and isocaseamembrin E, with IC 50 values of 0.25 µg/mL, 0.40 µg/mL, and 0.51 µg/mL, respectively. Cytotoxicity activity was considered slightly selective for pancreatic carcinoma (PANC-1). Structural determination was performed using NMR and mass spectrometry. This article is the first report of the isolation of these compounds from C. coriacea and their antiparasitic potentials. Due to the possible action of compound corymbulosine in apoptotic death, the compound could have its studies extended to cancer, as well. An interesting approach in this article is the fact that the authors have employed the zebrafish model as in vivo cytotoxicity assay rather than using mice model. Yet, the adjustment of the zebrafish model to test phytochemical compounds is also an open field.

Alkaloids: Potential Antimalarial Activity
Bioassay-guided fractionation of Hypoestes forskaolii (Vahl) R.Br. produced a phenanthroquinolizidine alkaloid, the isolated compound 15β-hydroxycryptopleurine-N-oxide (17), which has antiplasmodial activity. The chloroquine-resistant (K1) and chloroquinesensitive (FCR3) P. falciparum strains were very sensitive to the antimalarial activity of 15β-hydroxycryptopleurine-N oxide, with IC 50 values of 6.11 and 5.13 nM, respectively, while using chloroquine as the positive control [80]. A total of 15 mg was obtained from the plant, and a combination of techniques were used to purify the compound: silica gel column chromatography, high performance liquid chromatography (HPLC), and NMR (H, C, and two-dimensional).
From Cissampelos pareira (Linn) (Menispermaceae), a plant traditionally used to treat various diseases in India, a root ethyl acetate fraction with the most promising compound hayatinine (12), a bisbenzylisoquinoline alkaloid, was isolated (a total of 23 mg), having IC 50 values of 0.41 µM (INDO) and 0.509 µM (3D7) [81]. Chloroquine was used as the positive control. Silica gel column chromatography, HPLC, NMR (H, C, and two-dimensional), and liquid chromatography coupled to mass spectrometry (LC/MS) were utilized for this alkaloid.
The phytochemical investigation of Aconitum heterophyllum (Wall. ex Royle) (Ranunculaceae) roots led to the identification of 2-O-cinnamoyl hetisine, a diterpenoid alkaloid in the chloroform fraction of the plant [82]. Antiplasmodial in vitro studies indicated an IC 50 of 1.92 µM (INDO) and an IC 50 10.8 µM (3D7). The yield of this compound was very low: from 2.6 g of chloroform fraction, only 16 mg of this hetisine were obtained.
The tazopsine alkaloid (compound 10 in Figure 2), isolated from Strychnopsis thouarsii Baill. (Menispermaceae), an endemic Malagasy plant, showed antiplasmodial activity and an of IC 50, 7.88 µM against the NF54 strain, as well as a value of 4.07 µM against the 3D7 strain [83]. However, in P. yoelii infected mice, it was considered non-prophylactic, with 70% protection at a 100 mg/kg dose.

Terpenoids: Potential Antimalarial Activity
Sesquiterpene lactones have emerged as good starting points in the search for transmissionblocking drugs, with several studies demonstrating their potential. The most widely investigated member of this class of compounds is artemisinin, along with its derivatives. Greve and collaborators reported [84] that a triterpene derivative, compound rel-(16S,20S)dihydroxydammar-24-en-3-one (16), had the most promising antimalarial activity. For this triterpene derivative, silica gel column chromatography, HPLC, NMR (H, C, and two-dimensional), and LC/MS were used.
The dichloromethane extract obtained from Commiphora myrrha Engl. (Burseraceae), the oleo-gum resin, showed promising in vitro activity against P. falciparum NF54, with an IC 50 value of 1 µg/mL. Bioactivity-guided fractionation led to the isolation and characterization of 18 sesquiterpenoids. The sesquiterpene furanodienone (11) and the triterpene rel-(16S,20S)-dihydroxydammar-24-en-3-one (16) were the most active compounds found in this study, with IC 50 values of 7.4 and 2.8 µM, respectively, with chloroquine as the positive control [84]. The techniques employed to isolate the compounds were silica gel column chromatography, HPLC, flash chromatography, NMR (H, C, and two-dimensional), and LC/MS.
Phytochemical investigation of the n-buthanolic extract of the roots of Terminalia albida (Sc. Elliot) (Combretaceae) by Balde and collaborators [85] led to the isolation and identification of ten oleanane triterpenoids, of which albidic acid B (18), albidic acid C (19), and albidienic acid (20) showed moderate antiplasmodial activity, with IC 50 values ranging in the interval between 6 µM and 7 µM against K1 strain, also with chloroquine as the positive control. The isolation of the compounds was possible by employing the techniques of silica gel column chromatography, HPLC, NMR (H, C, and two-dimensional), and LC/MS.
Kim and colleagues [87] reported eight diterpenoids from the whole plant of Vitex rotundifolia L. f. (Verbenaceae). It is an important medicinal plant distributed in Korea, Japan, India, and China. The traditional uses of the plant comprise gastrointestinal diseases, chronic bronchitis, and migraine. The antiplasmodial activity was tested against the Dd2 strain, with an IC 50 of 1.2 µM for the compound named abieta-11(12)-ene-9β,13βendoperoxide, which was obtained from the n-hexane fraction (5.1 g), yielding a total of 15.4 mg. The β-peroxy bridge in this compound shows its importance for the antiplasmodial activity (Dd2), increasing it by up to 54 folds. Another promising isolated compound was Vitetrifolin E, with an IC 50 1.3 µM, which was isolated with a yield of 55.7 mg from 21 g of the methanolic fraction.
Two compounds isolated from the stem barks of Trichilia monadelpha (Thonn.) (J. J. De Wilde) (Meliaceae) were very active against the P. falciparum 3D7 strain: grandifotane A showed an IC 50 of 1.37 µM and khayanolide D an IC 50 of 1.68 µM, both against the 3D7 strain [88]. Both isolated compounds were obtained in the n-hexane/ethyl acetate fraction (150 mg for grandifotane A and 15 mg, both from initial 150 g of extract). The genus Trichilia comprises about 70 species distributed in tropical regions, mainly Africa and America, and the species T. monadelpha is used in traditional medicine for treating a variety of diseases, including depression, epilepsy, syphilis, and rheumatism [88].
Another genus of plants, Croton, is used in Tanzanian traditional medicine for the treatment of infections, malaria, tuberculosis, and cancer. In a recent study [89], stem bark and leaf extracts of Croton kilwae Radcl.-Sm. (Euphorbiaceae) endemic species (Tanzania and Mozambique) yielded the isolation of three new crotofolane diterpenoids with antiplasmodial activity at 50 µM concentration, and which were named as crotokilwaepoxides. Artesunate at 5 µM concentration was utilized as positive control and drug vehicle DMSO as negative control in parasitaemia assays. The structure of the antiplasmodial compounds was determined via NMR spectroscopic data and mass spectrometry, and one compound was identified as a new crotofolane 9-methoxycrotokilwaepoxide A.
Still, in the terpenoids class, dimeric sesquiterpenoids isolated and characterized from Sarcandra glabra subsp. Brachystachys (Blume) (Verdc.), a plant species of tropical biome in south Asia and a long-known folk medicine in China, may also look promising in terms of antimalarial properties. Zhou and collaborators have isolated and characterized many compounds of this chemical class from plants of Chinese folk medicine [90], and, recently, it was found that one of the isolated dimeric sesquiterpenoids showed an EC 50 value in the picomolar range (4.3 pM) [91], testing in vitro against P. falciparum Dd2 with DMSO as negative control. The in vitro compound has about 1000-fold potency compared to artemisinin. Therefore, this chemical class may be of particular interest in malaria drug discovery.
Du and collaborators [93] performed a bioassay-guided isolation of the methanol extract of Galtonia regalis (Hilliard) (B.L. Burtt) (Asparagaceae), and the extract exhibited antiplasmodial activity with IC 50 value less than 1.25 µg/mL. Two known cholestane glycosides and five new cholestane glycoside galtonosides were isolated after bioassayguided fractionation. Galtonoside B (15) showed the most potent antiplasmodial activity, with an IC 50 value of 214 nM against the multidrug-resistant Dd2 strain of P. falciparum. Silica gel column chromatography, Sephadex LH-20, HPLC, NMR (one-dimensional and two-dimensional), and MS/MS were used in combination for the isolation of the compound.
A biodereplication workflow was applied to an ethanolic extract of the medicinal plant Piper coruscans Kunth (Piperaceae) against the Plasmodium falciparum strain 3D7. This resulted in the isolation of two antimalarial chalcones, aurentiacin (7) and cardamonin (8), with IC 50 values of 2.25 and 5.5 µM, respectively [95]. The combination of NMR, MS/MS and LC/MS allowed isolation and characterization of the compounds.
In another study, the plant Aloe marlothii A.Berger (Xanthorrhoeaceae), found in southern African countries, and traditionally used in folk medicine to treat different diseases, was tested against asexual and sexual forms of P. falciparum parasites. Mianda and collaborators [97] isolated six different compounds of this plant, and the most promising compound identified was aloesaponarin I, with an IC 50 of 1.54 µg/mL (5 µM) against NF54 strain and IC 50 of 1.58 µg/mL (5 µM) against K1 strain.
In a recent study [98], a plant found in Asia, named Putranjiva roxburghii Wall. (Putranjivaceae), was found to contain the polyphenol quebrachitol, which had an IC 50 of 0.87 µg/mL and at a dosage of 30 mg/kg indicated a chemosuppression of 73.26% in a murine model (P. berghei K173). High selectivity index was obtained for this compound (SI = 158).
The species Paeonia officinalis L. (Paeoniaceae) belongs to a genus that comprises about 33 species. They are distributed in Europe and Asia, and 11 species are found in China. The roots of P. officinalis are used in traditional Chinese medicine due to its anti-inflammatory, analgesic, and sedative properties, as well as its antihypertensive and anti-ulcer properties. Through the combination of chromatographic techniques, including Sephadex LH-20, the compounds identified as methyl gallate and galloyl paeoniflorin were isolated and tested in vitro against two different strains of P. falciparum (D6 and W2) [48], which provided better IC 50 values against the W2 strain-0.61 and 2.91 µg/mL, respectively.
Antimalarial compounds from Mammea siamensis (Miq.) (T. Anders.) (Calophyllaceae) flowers, which were isolated via a bioassay-guided workflow, exhibited antimalarial activity. The result suggested that 1-hydroxy-5,6,7-trimethoxyxanthone (14) is a possible lead structure and a potent inhibitor of the Pf LDH enzyme. Negative cytotoxicity in Vero and HepG2 cells was observed [70]. Techniques, such as silica column gel, HPLC, and NMR (one-dimensional and two-dimensional) were employed to isolate the antimalarial structure.

Conclusions and Future Perspectives
The search for antimalarial compounds isolated from plant extracts remains challenging. The benefits of searching such compounds in flora are evident: the plethora of chemical structures potentially isolated from natural sources is still an underexplored space, and the diversity in the traditional use of endemic plants also reinforces the need for isolation of possible antimalarial compounds. However, different aspects in plant extracts and their isolated compounds are crucial to be understood: the possible synergisms that exist when combining plant extracts through different techniques, the presence of factors that interfere in the composition of the extracts such as seasonality that may interfere in the levels of plants secondary metabolites, and the empirical evidence originated from the traditional use.
This mini review has provided an overview of the last five years of the literature regarding plant extracts and their isolated natural compounds with potential antimalarial effects. Twenty chemical compounds isolated from plants with IC 50 < 10 µM against at least one P. falciparum strain were selected and discussed. Several rich antiplasmodial fractions derived from plant extracts have been reported in the literature. However, the link between traditional knowledge and further research in discovering potential natural products with pharmacological properties must be reinforced, and hyphenated techniques are crucial for better characterizing the chemical compounds structures and their targets.
In particular, a closer connection with the local communities and better documentation of the local scientific information gathered is needed to further aid natural products research. Some families, such as Annonaceae, Meliaceae, Asteraceae, and Euphorbiaceae, are rich sources of bioactive compounds capable of inhibiting Plasmodium in vitro. More specifically, the families Annonaceae and Asteraceae provide genera, such as Sphaerocoryn, Annickia, Petasites, and Sonchus, which could be further investigated for their antimalarial effects, based on the in vitro and in vivo experimental results, and the generation of potential lead compounds for drug development. Whether they have an appropriate cytotoxicity profile and whether they will have low IC 50 values in vivo should be investigated with the use of hyphenated techniques combined with omics-based approaches.
Author Contributions: G.d.J.G.R. and S.L.R.Y. performed the literature search, analyzed data, and wrote the manuscript, G.P. wrote and revised the manuscript, C.W. revised the manuscript and supervised the work. All authors have read and agreed to the published version of the manuscript.